Received 4 August 2009; revised 2 November 2009nopr.niscair.res.in/bitstream/123456789/7246/1/IJBB 46(6... · 2010-01-26 · enzymatic and non-enzymatic for prevention of overload

Indian Journal of Biochemistry & Biophysics

Vol. 46, December 2009, pp 421-440

Review

Oxidative Stress in Cardiovascular Disease

S V Vijaya Lakshmi1, G Padmaja

1, Periannan Kuppusamy

2 and Vijay Kumar Kutala

1

1Department of Clinical Pharmacology & Therapeutics, Nizam’s Institute of Medical Sciences, Hyderabad, India 2Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio, USA

Received 4 August 2009; revised 2 November 2009

Over the last two decades, it has become increasingly clear that reactive oxygen species (ROS), including free radicals

are involved in cardiovascular disease. In recent years, there has been a growing interest in the clinical implications of these

oxidants. The ROS are common by-products of many oxidative biochemical and physiological processes. They can be

released by xanthine oxidase, NAD(P)H oxidase, lipoxygenases, mitochondria, or the uncoupling of nitric oxide synthase in

vascular cells. ROS mediate various signaling pathways that underlie vascular inflammation in atherogenesis. Various

animal models of oxidative stress support that ROS have causal role in atherosclerosis and other cardiovascular diseases.

They are too reactive to be tolerated in living tissue, and aerobic organisms use sophisticated defense system, both

enzymatic and non-enzymatic for prevention of overload of free radicals. In a number of pathophysiological conditions, the

delicate equilibrium between free-radical production and antioxidant capability can be altered in favor of the former, thus

leading to oxidative stress and increased tissue injury. This review focuses on the biochemical evidences concerning

involvement of ROS in several cardiovascular diseases, namely atherosclerosis, heart failure, hypertension and

ischemia/reperfusion injury.

Keywords: Free radicals, Oxidative stress, Cardiovascular diseases, Antioxidants, Reactive oxygen species, Hypertension,

Blood pressure

Reactive oxygen species (ROS) participate in normal

cell signaling as mediators that regulate vascular

function1-5

. In the vascular wall, ROS are produced by

all layers, including endothelium, smooth muscle, and

adventitia6. ROS include superoxide anion radical (O2

-)

hydrogen peroxide (H2O2), hydroxyl radical (.OH), nitric

oxide (NO), and peroxynitrite (ONOO-) (Figure 1).

Under physiological conditions, ROS are produced in

low concentrations and act as a signaling molecule that

regulate vascular smooth muscle cell (VSMC)

contraction and relaxation, and participate in VSMC

growth7-9

. Under pathophysiological conditions, these

free radicals play important roles in various

conditions, including atherosclerosis, ischemia-

reperfusion injury, ischemic heart disease, arrhythmias,

cardiomyopathy, congestive heart failure, cancer, and

diabetes10-13

.

There is now considerable biochemical,

physiological and pharmacological data to support a

link between free radicals and cardiovascular tissue

injury. Major vascular risk factors, such as

hypertension, dyslipidemia, diabetes and smoking

are associated with a marked increase in vascular

ROS production. There is accumulating evidence,

suggesting that disease conditions are directly or

indirectly related to oxidative damage and that they

share a common mechanism of molecular and

cellular damage. As these mechanisms are

elucidated, it may be possible to improve the

techniques for clinical and pharmacological

intervention. The present review focuses on the

evidences concerning the involvement of free

radicals in cardiovascular diseases and their

relationship to specific pathophysiological events.

_____________

Author for correspondence

Tel: 614-292-8998; Fax: 614-292-8454

E-mail: [email protected]

Abbreviations: ACE, angiotensin converting enzyme; Angio-II,

angiotensin-II; AT1, angiotensin-II type 1 receptor; ARE,

antioxidant response elements; CVD, cardiovascular disease; ECs,

endothelial cells; EH, essential hypertension; ED, endothelial

dysfunction; GST, glutathione-S-transferase; H2O2, hydrogen

peroxide; IL-1β, interleukin-1β; LDL, low-density lipoprotein;

LOO., lipid hydroperoxyl radical; MCP1, monocyte chemotactic

protein-1; mtDNA, mitochondrial DNA; NO, nitric oxide; NOS,

nitric oxide synthase; NQ01, NAD(P)H: quinone oxidoreductase;

Nrf2, nuclear factor erythroid-2 related factor 2; O2-., superoxide;

OH, hydroxyl radical; ONOO-, peroxynitrite; OxLDL, oxidized

low-density lipoprotein; PDGF, platelet derived growth factor;

PKC, protein kinase C; ROCK, Rho-associated kinase; ROS,

reactive oxygen species; SOD, superoxide dismutase; TGF-β,

transforming growth factor-β; VSMC, vascular smooth muscle cell.

INDIAN J. BIOCHEM. BIOPHYS. VOL. 46, DECEMBER 2009

422

There are several potential sources of ROS

production. In cardiovascular disease (CVD);

the sources include xanthine oxidase14

,

cyclooxygenase15

, lipooxygenase16

, mitochondrial

respiration17,18

, cytochrome P45019

, uncoupled nitric

oxide synthases11,20,21

and NAD(P)H oxidase22

. They

have been identified as sources of ROS generation in

all types of vasculature. These sources may

contribute to ROS formation, depending on cell type,

cellular activation site and disease context.

Numerous studies have shown that various

physiological stimuli that contribute to pathogenesis

of vascular disease can induce the formation of ROS.

For example, a variety of agents, including

vasoactive agents such as angiotensin-II (Ang II),

endothelin-1 and thrombin have been shown to

activate NAD(P)H oxidase23

. Treatment of VSMCs

with Ang II causes an increase in expression of

NAD(P)H oxidase as well as ROS production24,25

.

Cytokines (IL-1β, TNF-α), growth factors (platelet-

derived growth factor, PDGF; transforming growth

factor-β, TGF-β) and hemodynamic forces (shear

stress and cyclic stretch) can regulate the expression

and/or activity of the vascular NAD(P)H oxidase26-30

.

Recent studies suggest that intracellular ROS

production may also be derived from the

mitochondria. The production of mitochondrial

superoxide radicals occurs primarily at two discrete

points in the electron-transport chain, namely at

complex I (NADH dehydrogenase) and at complex

III (ubiquinone-cytochrome c reductase)31

.

Several intracellular signal events stimulated by

ROS have been defined, including the two members

of mitogen-activated protein-kinase family (ERK1/2

and big MAP kinase, BMK1), tyrosine kinases (Src

and Syk) and different isoenzymes of PKC as redox-

sensitive kinases32-36

. ROS regulation of signal

transduction components include the modification in

the activity of transcriptional factors such as NF-κB

and others that result in changes in gene expression

and modifications in cellular responses. The small

guanosine triphosphatase (GTPase) Rho works as a

switch and plays an important role in various cellular

physiologic functions, including actomyosin-based

cellular processes such as cell adhesion, migration,

motility, cytokinesis and contraction, all of which

may be involved in the pathogenesis of

atherosclerosis37

.

Figure 1Sources of reactive oxygen species (ROS) generation and their effects on signaling systems in cardiovascular disease

[Activated NAD(P)H oxidases, lipoxygeneases and xanthine oxidase generate O2-., NOS switches from a coupled state to an uncoupled

state and generates O2-. with decreased 5,6,7,8-tetrahydrobiopterin (BH4) or L-arginine. Dysfunctional mitochondrial electron-transport

chain is another source of O2-.. ROS reduce bioavailability of NO, leading to endothelial dysfunction. ROS influence the activity of a

variety of cellular signaling pathways ultimately leading the changes in the expression of redox-sensitive genes, which regulate cellular

process involved in cellular apoptosis/death that may involved in the pathogenesis of cardiovascular disease]

VIJAYA LAKSHMI et al.: OXIDATIVE STRESS IN CARDIOVASCULAR DISEASE

423

There is growing evidence that Rho-associated

kinase (ROCK) (also known as Rho-kinase), the

immediate downstream target of the small GTP-

binding protein Rho contributes to endothelial

dysfunction (ED) and vascular disease38-42

. The

clinical evidence has demonstrated that ROCK is

significantly activated in patients with coronary

vasospasm43

, hypertension44

, stable-effort angina45

and in current smoking in healthy subjects46,47

.

Evidences indicate that ROCK is significantly

associated with the regulation of not only endothelial

nitric oxide synthase (eNOS) expression, but also

eNOS phosphorylation, both of which are important

mechanisms for regulating endothelial function and

subsequent cardiovascular injury38-42,48

. A significant

relationship has been found between ED and

increased ROCK activity in smokers47

. The aging and

cigarette-smoking are also involved in increase in

ROCK activity, which may be partly explained by the

significant correlation between ROCK and endothelial

function. These observations suggest that activation of

ROCK is involved in several aspects of the

atherosclerotic process, including ED44

.

Nitric oxide (NO) has recently emerged as an

important mediator of cellular and molecular events

that impact the pathophysiology of myocardial

ischemia. NO produced by vascular endothelium is

shown to possess potent vasodilatory properties and

also an inhibitor of platelet aggregation which may be

beneficial to the early stages of focal myocardial

ischemia49

. It may also facilitate collateral blood flow

to the ischemic territory44

. An increase in intracellular

Ca2+

(resulting from the activation of voltage-gated

Ca2+

channels or ligand-gated Ca2+

channels or from

the mobilization of intracellular Ca2+

stores) could

activate the enzyme NO synthase44

, which catalyzes

the synthesis of NO from L-arginine and molecular

oxygen. NO may cause cytotoxicity through

formation of iron-NO complexes with several

enzymes including mitochondrial electron-transport

chain, oxidation of protein sulfhydryls and DNA

nitration44

. It may also mediate cell death through

formation of the potent oxidant peroxynitrite (ONOO-),

the reaction product of NO with O2•-50

. Peroxynitrite

decomposes to the hydroxyl free radical (•OH) and to

radical nitrogen dioxide (NO2•), which are potent

activator of lipid peroxidation.

The Nrf2 (NF-E2-related factor-2)/antioxidant

response element (ARE) pathway is a cis-acting

sequence that mediates transcriptional activation of

genes in cells exposed to oxidative stress51-53

. The

ARE is present in the 5' flanking regions of genes

encoding phase II detoxification enzymes and cellular

antioxidant proteins including glutathione-S-

transferase (GST)54

, NAD(P)H: quinone oxido-

reductase (NQ01)55

, glucuronosyl transferase54

,

heme oxygenase-156

and ferritin57

. Nrf2 is the

transcription factor that upon activation by

oxidative stress binds to the ARE and activates

transcription of ARE-regulated genes58,59

. In

vascular cells, the ARE pathway is activated by

oxLDL60,61

and NO exposure62

. ECs when exposed

to prolonged laminar flow show a marked increase

in the expression of ARE-mediated genes such as

GST, NQO1, HO-1 and ferritin through Nrf2-

dependent mechanism63

.

Protective pathways in mammalian cells and tissues

To prevent overloading of free radicals and

peroxides, aerobic organisms use a sophisticated

defense system, which operates both in intra- and

extracellular aqueous phases and in membranes.

Antioxidant defense strategies are committed to

counteract the oxidative attack in its early

moments, i.e. formation of primary radicals, as well

as during the initiation and chain-propagation

processes. Antioxidant protection can be viewed as

consisting of four sequential levels of defensive

activity: preventive, chain-breaking, repairing, and

adaptive. The first level of defense, which is largely

enzymatic, involving enzymes such as superoxide

dismutases (SODs), glutathione peroxidases (GPx)

and catalase is concerned with the control of

formation and proliferation of primary radical

species derived from molecular oxygen. There are

three different forms of SODs (manganese,

copper/zinc, extracellular) that metabolize O2-. to

hydrogen peroxide (H2O2). Catalase and at least

four isoforms of GPx then convert H2O2 into water.

Extracellular SOD (ecSOD) is produced by VSMCs

and not endothelial cells64,65

and localizes in highest

concentrations between the endothelium and

VSMCs66

. The second level of defense, which

involves vitamins C and E and probably

carotenoids is concerned with the prevention of

proliferation of secondary radicals in chain

reactions, such as lipid peroxidation, initiated and

driven by primary radicals. The third level of

defense is the enzymatic prevention of formation of

secondary radicals from chain-terminated

derivatives and enabling the removal of such


424

molecules from an environment in which metal-

catalyzed reactions might cause further oxidative

damage.

Free radicals in cardiovascular diseases

Oxidative stress and endothelial dysfunction

Endothelium is the bioactive inner layer of the

blood vessels, which serves as an important locus on

control of vascular and thus other organ functions

regulating vascular tone permeability67

. It produces

components of extracellular matrix such as collagen

and a variety of regulatory mediators, including NO,

prostanoids, endothelin-1 (ET-1), angiotensin II

(Ang II), tissue-type plasminogen activator (t-PA),

von Willebrand factor (vWF), adhesion molecules

and cytokines. Endothelial dysfunction (ED) is an

early event in atherosclerotic disease, preceding

clinical manifestations and complications. Evidences

have shown that ED is a strong predictor of future

cardiovascular events in patients with cardiovascular

risk factors68

. ROS have been implicated as

important mechanisms that contribute to ED and

may function as intracellular messengers that

modulate signaling pathways. Increased ROS

production is a major cause of ED in experimental

and clinical atherosclerosis69,70

.

Among important molecules synthesized by

endothelial cells is NO, which is a potent

vasodilator49

. The important functions of NO include

anti-platelet and anti-proliferative, permeability-

decreasing and anti-inflammatory properties71

. NO

Inhibits leukocyte adhesion and rolling as well as

cytokine-induced expression of VCAM-1 (vascular

endothelial cell adhesion molecule) and MCP-1

(monocyte chemotactic protein)72

, effects partly

attributable to inhibition of the transcription factor

NF-κB73

. ED leads to a rapid decrease in NO

production or availability, partly due to inactivation of

NO by superoxide.

Superoxide reacts rapidly with NO, resulting in

the formation of peroxynitrite and loss of NO

bioavailability. Decrease in NO bioavailability

could also result from reduced expression or

activity of eNOS, increased generation of asymmetric

dimethylarginine (ADMA; an endogenous circulating

inhibitor of NOS), decreased availability of 6R-

tetrahydrobiopterin (BH4; an essential NOS

cofactor) or increased inactivation of NO by

superoxide74

. Recent studies have shown that ROS,

in particular peroxynitrite can oxidize

tetrahydrobiopterin27,75

. Polymorphisms have been

observed in a variety of genes whose products have

been implicated in ED. In NOS3 (eNOS) gene,

more than 15 polymorphisms exist in the promoter

region that might influence reduced gene

expression. The presence of polymorphisms in

other genes include methylene tetrahydrofolate

reductase, angiotensin-converting enzyme, p22phox

,

glutathione-S-transferase and cytochrome P450,

which can cause ED76

.

ROS are involved in the endothelial and VSMC

pro-inflammatory signaling, particularly in the

regulation of VCAM-1 and MCP-1 expression.

They also are involved in redox-signaling cascade,

leading to vascular pro-inflammatory and pro-

thrombotic gene expression involving the

transcription factor NF-κB. Finally, ROS activate

matrix metallo-proteinases (MMPs), contributing to

plaque instability and rupture77

. Homocysteine

(Hcy) and oxidized LDL (oxLDL) have been shown

to enhance the activity and expression of oxidative

stress markers, such as NF-κB and heme

oxygenase-178

. These results suggest that these pro-

atherogenic stimuli increase oxidative stress in

endothelial cells and thus explain the loss of

endothelial function associated with the atherogenic

process. ED in experimental atherosclerosis could

be reversed by administration of superoxide

scavengers, suggesting that increased vascular

superoxide production represents a major cause of

ED75,79

. Tetrahydrobiopterin improves endothelial

dysfunction and vascular oxidative stress in

microvessels of intrauterine undernourished rats80

.

Oxidative stress and atherosclerosis

Atherosclerosis originates from ED and

inflammation. The importance of oxidative stress in

the development of atherosclerosis seems to be

widely accepted. The free radicals are involved

throughout the atherogenic process, beginning from

ED in an otherwise intact vessel wall up to the

rupture of a lipid-rich atherosclerotic plaque,

leading to acute myocardial infarction or sudden

death81

. The development of atherosclerosis is a

multi-factorial process in which both elevated

plasma cholesterol levels and proliferation of

smooth muscle cells play a central role.

Atherogenesis is an alteration of the artery wall that

includes two major phases: (i) adhesion of

monocytes to the endothelium and their migration


425

into the sub-endothelial space and differentiation

into macrophages. These cells ingest (oxidized) low

density lipoproteins (LDL) and through this process

they are transformed into "foam cell"; (ii) VSMC

migration from the media into the intima and their

proliferation with the formation of atherosclerotic

plaque.

Oxidation of low-density lipoprotein

Considerable in vivo evidence from animal and

human studies support the important role of oxygen

free-radical reactions in atherogenesis and

atherosclerotic coronary heart disease81-84

. While the

exact mechanisms for atherogenesis are not

completely understood, recent studies suggest that

oxidative modification of low-density lipoproteins

(LDL) is a critical factor85-87

. Thus, LDL is the "bad

actor" in the free-radical hypothesis of atherosclerosis

and may be oxidatively modified by all major cell

types of the arterial wall, including endothelial cells,

smooth muscle cells and macrophages via their

extracellular release of ROS. Hydroxyl radicals may

initiate the peroxidation of long-chain polyunsaturated

fatty acids within LDL molecule, giving rise to

conjugated dienes and lipid hydroperoxy radicals

(LOO•). This process is self-propagating, such that

LOO• can attack adjacent fatty acids until complete

fatty acid chain fragmentation occurs. A number of

highly reactive products, including malondialdehyde

and lysophosphatides then accumulate in the LDL

particle. These products interact with the amino side

chain of the apoprotein B 100 and modify it to form

new epitopes that are not recognized by the LDL

receptor.

Oxidatively modified LDL (OxLDL) is avidly

taken up by sub-endothelial macrophages via the

"scavenger" receptor pathway which does not

recognize native, unmodified LDL. Through the

scavenger receptor, unlimited amounts of modified

LDL are ingested by the monocyte/macrophage,

which is now a "foam cell" in the arterial intima.

Accumulation of LDL-laden foam cells beneath

arterial endothelium results in the formation of "fatty

streak", the earliest histopathological evidence of the

development of atherosclerotic plaque88,89

. Oxidized

LDL also stimulates the release of monocyte-derived

TNF-α and IL-1β, leading to smooth muscle cell

proliferation. Elaboration of collagen and elastin by

smooth muscle cells leads to plaque formation and

fibrosis88

. Lipid peroxides also inhibit synthesis of

prostacyclin, an antiplatelet-aggregation substance,

which can result in platelet adherence and

aggregation. Platelets release growth factors,

subsequently leading to smooth muscle cell

proliferation and migration to intima. Besides, this

may also lead to thrombosis due to the aggregation

of platelets88

.

OxLDL has additional atherogenic and many pro-

inflammatory properties. It stimulates the expression

of macrophage colony-stimulating factor (M-CSF),

granulocyte macrophage colony-stimulating factor

(GM-CSF) and monocyte chemotactic protein-1

(MCP-1) by endothelial cells and is also cytotoxic to

these cells90,91

. oxLDL is chemotactic for monocytes

and inhibits the motility of macrophages. It is highly

immunogenic, forming immune complexes in the

arterial wall that can also be taken by macrophages.

Antibodies against oxidized LDL have been detected

in rabbit atherosclerotic lesions, and the plasma of

rabbits and humans contains autoantibodies that react

with several forms of oxidized LDL92

. Atherosclerotic

lesions from human aorta contain lipid peroxides, and

the peroxide content correlates with the extent of

atheroma93

. Detectable levels of oxLDL are also

found in human plasma, and elevated plasma peroxide

levels have been found in diabetics, smokers and

patients with coronary disease92,94,95

.

Oxidative stress and mitochondrial dysfunction

The mitochondria are shown to be sensitive to

both ROS-mediated damage and alterations in

function96-101

. Recent studies have shown that

intracellular ROS production may also be derived

from the mitochondria. The production of

mitochondrial ROS occurs primarily at two discrete

sites in the electron-transport chain, namely at

complex I (NADH dehydrogenase) and at complex III

(ubiquinone-cytochrome c reductase). Under patho-

physiological conditions, the electron-transport chain

may become uncoupled, leading to increase in O2-.

production102

. Mitochondrial DNA (mtDNA) is

particularly susceptible to modification by ROS/RNS

(reactive nitrogen species) because (i) mtDNA is in

close proximity to the site of ROS/RNS production,

(ii) mtDNA lacks histone proteins, which can protect

it from oxidative damage103

, and (iii) poor DNA

damage-repair activity104

. Damage to mtDNA can

lead to functional changes in the cell, as it encodes

several critical protein components of the

mitochondrial respiratory chain.

Numerous studies have reported the existence of a

correlation between DNA damage and


426

atherosclerosis105,106

. The patients with CVD have

shown increased mtDNA damage when compared

with healthy controls in the both the heart and

aorta18,105,107,108

. DNA adduct levels are significantly

higher in the thoracic aorta from atherosclerotic

patients compared to controls109

. Increased

immunoreactivity against 8-oxoG (a product of

oxidative DNA damage) in plaques of the human

carotid artery compared with the adjacent inner media

has been reported109

. Multi-variate analysis reveals

that DNA-adduct levels are a significant predictor of

stage of atherosclerosis110

. Consistent with these

studies is the observation that mtDNA damage is

increased in vascular tissues in CVD patients18

. Pro-

atherogenic risk factors such as smoking,

hypercholesterolemia and obesity are all associated

with increased mtDNA damage108

. The exogenous

ROS-mediated mtDNA damage is reported to

decrease mtDNA-encoded gene transcription in a

dose-dependent manner and the extent of

atherosclerosis correlates well with mtDNA damage

in the aorta from humans and mice18

.

Atherogenesis is also considered a chronic

inflammatory disease of the vessel wall111

. Thus, in

this condition, after exposure to endotoxins and

certain cytokines (TNF-α, IL-1β, expression of iNOS

in vascular endothelial cells, smooth muscle cells,

endocardium and macrophages located within the

vessel wall leads to prolonged synthesis of large

amounts of NO and also to the endothelial cell

damage or dysfunction. The initial hypothesis for

deleterious effects of NO has been based on its free-

radical nature and its reactivity. NO diffuses out and

can reach adjacent cells, where it reacts with the iron-

sulfur centers of several important enzymes from the

mitochondrial electron-transport chain and/or

ribonucleotide reductase, the enzyme necessary for

DNA synthesis99,100

. Recently, it has been suggested

that iNOS is expressed in aneurysmal atherosclerotic

human aorta112

. Besides, studies with NO-donor drugs

suggest that overproduction of NO in the human heart

might decrease contractility and impair diastolic

relaxation. Based on these findings, it may be

proposed that the net effect of NO modulation in

cardiovascular system probably results from a balance

between beneficial hemodynamic effects and

cytotoxicity. It remains to be determined, why normal

physiological production of NO is protective in

cardiovascular system and may prevent atheroma

formation, whereas overproduction of NO after iNOS

induction is potentially harmful. Taken together, these

studies indicate that ROS are clearly associated with

enhanced susceptibility to atherosclerosis.

Oxidative stress in hypertension

ROS and oxLDL may play a critical role in the

pathophysiology of hypertension. The studies in

experimental hypertension and hypertension in

humans have demonstrated increased generation of

ROS13,113

. Inactivation of the genes for the enzymes

that generate ROS in mice results in lowering of their

blood pressure. Antioxidants may reduce blood

pressure in animal models of hypertension and

prevent target organ damage12

. They also demonstrate

some beneficial effects in essential hypertension (EH)

in humans114-116

. The reports suggest that EH is

associated with increased superoxide anion and H2O2

production, as well as decreased antioxidant

capacity26-28

. The involvement of reactive oxygen

intermediates in EH is also suggested by the increased

level of lipid peroxides and decreased concentrations

of antioxidant vitamin E in plasma of essential

hypertensive patients117

.

The underlying mechanism that leads to the

oxidative stress in EH remains largely unexplored.

Reactive oxygen radicals may play a dual role in EH.

On one hand, they may inactivate NO by converting

them into peroxynitrite in reaction with superoxide

anion, thereby causing arteriolar vasoconstriction

and elevation of peripheral hemodynamic

resistance118

. On the other hand, enhanced

production of free radicals may serve as trigger

mechanism for oxidative damage of numerous

macromolecules, for example, LDL. The enhanced

LDL oxidation has been observed in EH patients 119,120

. This conclusion is based on findings obtained

in isolated LDL (which appears more prone to

oxidation triggered by exogenous stimuli) and on

demonstration of autoantibodies directed against

epitopes generated during oxidative modification of

apoprotein B-100119

.

To understand the mechanism for oxygen free

radical formation in hypertension, the cellular source

must be identified. The endothelial cell, which is

recognized as a source of NO has also been identified

as a potential site of ROS production12,121

. Superoxide

radicals in and around vascular endothelial cells play

a critical role in the pathogenesis of hypertension.

Increased superoxide anion and H2O2 production by

leukocytes isolated from hypertensive patients


427

(Figure 2) and increased levels of lipid peroxides

(Figure 3A) and decreased levels of nitrites in plasma

have been observed (Figure 3B)121,122

. Besides, the

spontaneously hypertensive rats have shown an

elevated number of circulating leukocytes that

produce superoxide compared with normotensive

control123

. Essential hypertensive patients have been

shown to produce excessive amounts of ROS124,125

and decreased antioxidant capacity126

. Activation of

renin-angiotensin system is a major mediator of

NAD(P)H oxidase activation and ROS production in

human hypertension127

.

The molecular basis of hypertension is complex;

more than 50 genes have been implicated in the

regulation of blood pressure26

. The role of AT1 receptor

in regulating hypertension has been investigated in both

in vitro and animal models. Ang II modulates

hypertension through its effects on the renin-angiotensin

system and the stimulation of NAD(P)H oxidase in

vascular walls. It also directly regulates NAD(P)H

oxidase activation by enhancing a rapid translocation of

small GTPase rac 1 to the cell membrane29

or by

phosphorylating and translocating p47phox

membrane

translocation to cell membranes30

. The most important

source of ROS in blood vessels appears to be NAD(P)H

oxidase1,12

. a multi-subunit enzyme6,22

having NAD(P)H

as electron donor. The best characterized NAD(P)H

oxidase is found in phagocytes, neutrophils, monocytes

and macrophages128

.

Non-phagocytic oxidase is the main source of ROS

in blood vessels6,129

, present in the endothelium130

and

VSMCs in the media129,130

. Transgenic mice that

express constitutively active rac1 in VSMCs exhibit a

hypertensive phenotype; increased ROS production

and treatment with antioxidants reverses

hypertension131

. The activity of vascular NAD(P)H

oxidase is modulated by many different factors that

include cytokines, growth factors and vasoactive

peptides. Stretch, pulsatile strain and shear stress may

activate NAD(P)H oxidase6,132

. Ang II not only

stimulates NAD(P)H oxidase, but also enhances the

expression of the subunits of NAD(P)H oxidase. It

induces ROS generation by endothelial cells, VSMCs

and adventitial fibroblasts via stimulation of AT1

receptors133

. PDGF, TGF-β, TNF-α and thrombin also

activate NAD(P)H oxidase in VSMCs134-136

.

Figure 2—Rate of superoxide and hydrogen peroxide generation

in polymorphonuclear leukocytes (PMNLs) of hypertensive rats

[(A) Rate of superoxide generation and (B) rate of hydrogen

peroxide generation by PMNLs from Control (n = 18), untreated

hypertensive (n= 30), and post-treated hypertensive (n = 18) rats.

PMNLs (1 x 106 cells/ml) were incubated with and without

phorbolmyristate acetate (PMA, 200 ng/ml), nitroblue tetrazolium

(NBT, 1%) in PBS (pH 7.4) for 20 min at 37oC and the blue color

formed was read at 560 nm117. Hydrogen peroxide generated by

PMNLs was estimated by horseradish peroxidase method by

incubating PMNLs with 1% phenol red containing peroxidase

(3.75 U/assay) and the absorbance was read 610 nm209. All values

are expressed as mean ± SD. *p< 0.05 vs control; **p< 0.05 vs

untreated hypertension (Adapted from117)]

Figure 3—Plasma levels of malondialdehyde (MDA) and nitrite in

hypertensive rats [(A) Malondialdehyde (MDA), (B) nitrite (end

product of NO) in control (n=18), untreated hypertensive (n = 25),

and post-treated hypertensive (n = 18) rats. The amount of lipid

peroxidation products (MDA) was determined by thiobarbituric

acid (TBA) method and measured at 532 nm117,210. Plasma nitrite

level was measured by using Greiss reagent (1% sulfanilamide in

5% H3PO4 + 0.1% naphthalene-ethylenediamine dihydrochloride)

at 543 nm211. All values are expressed as mean ± SD. *p< 0.05 vs

control; **p< 0.05 vs untreated hypertension (Adapted from117)]


428

Endothelin-1 increases NAD(P)H oxidase activity in

human endothelial cells via ETA receptors119

. The

statins, antihypertensive drugs, such as β-blockers,

calcium channel blockers, angiotensin-converting

enzyme (ACE) inhibitors and angiotensin receptor

blockers decrease the expression of NAD(P)H

oxidase subunits and its activity137-140

.

The antihypertensive action of ACE inhibitors and

angiotensin receptor blockers might be due to

inhibition of NAD(P)H oxidase activity and decreased

ROS production127

. The beneficial effects of

β-adrenergic blockers (carvedilol) and some calcium

channel blockers may be mediated, in part by

decreasing vascular oxidative stress139,144

. The genetic

models of hypertension, such as SHR (spontaneously

hypertensive rat) and stroke-prone SHR exhibit

enhanced NAD(P)H oxidase-mediated superoxide

generation by increased expression of its subunits

(p22phox

and p47phox

) in conduit (aorta) and resistance

arteries (mesenteric) and in the kidney113,141

.

Polymorphisms have been identified in the promoter

region of the p22phox

gene in SHR, which could

contribute to increased NAD(P)H oxidase activity142

.

Treatment with antioxidant vitamins, NAD(P)H

oxidase inhibitors, SOD blockers, folic acid AT1

receptor blockers decrease vascular superoxide

production, lipid-peroxidation products and may

decrease development of blood pressure elevation in

genetic hypertension113,116,143

.

Polymorphisms in p22phox

gene may play a role in

altered NAD(P)H oxidase-generation of superoxide in

humans, particularly 903(A/G) polymorphism142

.

However, in homozygous individuals with the T allele

of the C242T CYBA, polymorphism may have

reduced vascular oxidative stress145

. Taken together,

these observations strongly suggest that oxidative

stress is a modulator of hypertension, a potential risk

factor for atherosclerosis.

Oxidative stress and heart failure

Oxidative stress is increased in heart failure and

may contribute to many of the structural and

functional changes that characterize disease

progression. There are both indirect and direct

evidences of increased oxidative stress in humans

with heart failure. In patients with heart failure, the

level of the lipid peroxidation product

malondialdehyde (MDA) is increased in plasma.

Direct evidence of increased myocardial oxidative

stress is also obtained from the fact that the level of 8-

iso-prostaglandin F2α (8-isoprostane) is increased in

the pericardial fluid of the patients with heart failure.

The involvement of oxidative stress in heart failure is

further supported by the prevention of the progression

of several pathological processes such as cardiac

hypertrophy, cardiac myocyte apoptosis, ischemia-

reperfusion and myocardial stunning, which can lead

heart failure in animals models and TNF-α and Ang

II-induced hypertrophy in cardiac myocytes is

prevented by vitamin E, hydroxyanisole and

catalase146

. Overexpression of catalase significantly

reduces Ang II-induced hypertrophy and transfection

of antisense p22phox

inhibits Ang II-induced H2O2

production. This suggests that NAD(P)H oxidase-

induced oxidative stress leads to the hypertrophy9.

There are a number of potential sources of ROS in

the myocardium. Several enzyme systems generate

superoxide and among these, the mitochondria

appear to be an important source of myocardial ROS

in the failing heart. A small fraction of the electrons

that pass through the mitochondrial electron

transport chain may 'leak', thereby reacting with

molecular oxygen to form superoxide. Electron

paramagnetic resonance (EPR) spectroscopy with a

superoxide spin-trap shows 2.8-fold increase in ROS

in mitochondria from failing hearts, together with a

decrease in the activity of electron transport complex

I, suggesting that in heart failure, a functional

uncoupling of the mitochondria contributes to

increased ROS formation. Increased production of

ROS may decrease NO bioavailability and impair

diastolic function147

. In addition, increased

peroxynitrite may cause cytokine-induced

myocardial contractile failure by inactivating

sarcoplasmic Ca2+

-ATPase and dysregulating Ca2+

homeostasis148,149

.

Evidence also suggests that oxidases may

contribute to ROS generation in the myocardium.

Xanthine oxidase activity is increased in the failing

heart and xanthine oxidase inhibitors improve

myocardial energetics in a dog model of heart failure

and in humans with heart failure150

. NADPH oxidase,

a plasmalemmal enzyme that generates superoxide in

the cytosol is another oxidase implicated in

myocardial failure. In the failing myocardium of

patients with ischemic or dilated cardiomyopathy,

NAD(P)H oxidase-derived ROS are upregulated. In

patients with heart failure, plasma TNF-α and platelet-

derived NAD(P)H oxidase activity is also elevated151

.

In the failing myocardium, the translocation of

regulatory p47phox

from the cytosol to the sarcolemmal


429

membrane is recently demonstrated152

. Together,

these results suggest that oxidative stress has a role in

the pathophysiological cardiac dysfunction in heart

failure.

Oxidative stress and ischemia-reperfusion myocardial injury

Exposure of myocardial tissue to a brief transient

ischemia, followed by reperfusion has attracted

remarkable attention in recent years. Myocardial

ischemia occurs when myocardial oxygen demand

exceeds oxygen supply. Unless reversed, this

situation results in cell injury, leading to clinical

myocardial infarction. Reperfusion of ischemic

myocardium is recognized as potentially beneficial,

because mortality is directly proportional to infarct

size, and the severity and duration of ischemia.

Reperfusion of ischemic myocardium can restore

oxygen and substrates to the ischemic myocardial

cells, but this process may create another form of

myocardial damage termed "reperfusion

injury"153,154

. Thus, restoration of a normal blood

flow in the heart by methods, such as angioplasty,

thrombolysis cardiopulmonary bypass can lead to

specific lesions (arrhythmias, deficit in contractility,

necrosis), the importance of which also depends on

the duration of ischemia.

The damage to the myocardial cell induced by

cycles of ischemia and reperfusion may be due, in

part to the generation of toxic ROS such as

superoxide radical, H2O2 and hydroxyl radicals154-157

.

The active involvement of free radicals in the

ischemia-reperfusion damage is demonstrated by

direct and indirect experimental evidences. Direct

evidence arises from the possibility of measuring

radicals in myocardial tissue by EPR spin-trapping21

;

indirect evidence by the measurement of the

products of free radical attack on biological

substrates (usually MDA as a measure of lipid

peroxidation) and intracellular and extracellular

antioxidant capacity23

.

EPR spectroscopy has shown an increased free-

radical production in blood after reperfusion of infarct

tissue21

. EPR signals are also recorded in blood

samples taken from coronary sinus of patients

undergoing percutaneous transluminal coronary

angioplasty (PTCA), an ideal model of myocardial

ischemia-reperfusion21

. In patients undergoing

cardiopulmonary bypass, an increased free-radical

generation and reduction of blood-antioxidant

capacity in plasma have been reported, following

aortic declamping23

. Furthermore, the experimental

findings suggest an impairment of antioxidant

mechanisms in the ischemic tissue23

. Evidence to

support this is also obtained from the cardioprotective

effect of agents capable of inducing antioxidant

enzymes in the heart and from the beneficial effects of

several enzymatic free-radical scavengers,

antioxidants and iron chelators in reperfused

myocardium158

.

Reperfusion of the isolated rat heart with

oxygenated buffer has been shown to generate free

radicals, as detected by EPR spectroscopy159,160

. A

burst of free-radical generation, such as of .OH

., R

.

and RO. adducts of 5,5,-dimethyl-1-pyrroline-N-oxide

(DMPO) is observed during early period of

reperfusion, with peaking occurring within 30 sec of

reperfusion (Figure 4). The reperfusion-induced ROS

generation is markedly decreased by SOD, suggesting

that adduct formed is from ROS. In an another

study, pretreatment of heart with the plant-based

antioxidants Spirulina and C-phycocyanin significantly

attenuates the I/R-induced ROS generation (Figure 5)161

.

Figure 4—EPR spectra of effluent of heart perfusate [(A) pre-

ischemia, (B) effluent collected 2 min after reperfusion with

DMPO (40 mM), showing hydroxyl adduct and alkyl adduct,

(C) simulation of DMPO-OH adduct, (D) simulation of

DMPO-alkyl adduct, and (E) C + D simulation of both

DMPO-OH adduct + stimulation of DMPO-alkyl adduct

(compare with 4B)]


430

The oxygen tension during the initial reperfusion of

ischemic myocardium may modulate early ROS

generation162

. In hearts reperfused with 2% O2 for

the first 5 min, followed by 95% O2, ROS

generation peaks in the first 2 min of reperfusion

(Figure 6) Furthermore, the magnitude of ROS

generation is significantly higher in the 2% O2

group, when compared to the 95% O2 or 21% O2

(Figure 6).

The failure of all energy-dependent mechanisms

leads to deterioration of membrane ion gradients,

opening of selective and unselective ion channels and

equilibration of most intracellular and extracellular

ions. As a consequence of this "anoxic

depolarization", K+ ions leave the cell, while Na

+ and

Ca2+

ions enter. Cellular accumulation of ions causes

formation of cytotoxic edema. Intracellular Ca2+

overload can also set off a cascade of events which

may lead to the formation of ROS. The elevated Ca2+

concentration activates proteases that can convert

xanthine dehydrogenase to xanthine oxidase. During

reoxygenation, xanthine oxidase can use O2 as an

electron acceptor, leading to formation of superoxide

anion and H2O2, which can react to produce •OH

radicals. These reactive species are responsible for the

tissue damage. ROS produced from xanthine oxidase

play a major role in causing tissue damage in

ischemia-reperfusion injury, as evidenced by the

ability of inhibitors of xanthine oxidase in protecting

against such damage in experimental models of

myocardial infarction163

.

Another source of ROS generation is the intra-

mitochondrial electron-transport chain. Free radicals

produced in mitochondria may also cause point

mutations, DNA cross-link and DNA strand breaks in

mitochondrial genes. The damage to mitochondrial

genome results in impaired respiration, increasing

further the possibility of oxygen-radical production.

Impaired mitochondrial function and increased

production of superoxide are very common

reperfusion-associated events. The activities of

components of mitochondrial respiratory chain are

markedly reduced during post-ischemic reperfusion or

post-hypoxic reoxygenation164

. Experimental studies

suggest that mitochondrial dysfunction results in

increased production of superoxide by this organelle

after exposure of cardiac muscle to ischemia-

reperfusion165

.

Figure 5—Myocardial oxygen free radical formation at

reperfusion with antioxidants, Spirulina (SP) and C-phycocyanin

(PC) [(A) Time course and (B) Quantitation of 5,5,-dimethyl-1-

pyrroline-N-oxide (DMPO) adducts of radicals at 1 min of

reperfusion. Free radical generation was measured using spin-trap

(DMPO) with and without SP and PC in hearts subjected to

30 min ischemia and 45 min of reperfusion. DMPO (40 mM final

concentration) was infused through sidearm during reperfusion,

coronary effluent was collected at 0.5 – 10 min, and DMPO

adduct formation was measured by electron spin resonance

spectroscopy. Values are mean ± SD from 3 independent

experiments. *p < 0.05 vs control (Adapted from161)

Figure 6—Myocardial oxygen free radical formation at

reperfusion with different O2 concentrations [The free radicals

were measured as DMPO adducts in the effluent using spin-

trapping EPR spectroscopy. The DMPO adduct peaked in the first

2 min in all groups and was significantly elevated in the most

hypoxic reperfusion group (2% O2) compared with the 20% and

95% O2 reperfusion groups (*p < 0.001 versus 95% O2 and 20%

O2, n=4/group). (Adapted from162)]


431

Reperfusion after an ischemia period is associated

with impaired bioavailability of NO, most likely due

to enhanced inactivation of NO by superoxide and

reduced production of NO. Several studies on NO and

NOS inhibition in models of I/R have yielded mixed

results. Studies on isolated rat heart have shown that

L-arginine and several NO donors can attenuate post-

ischemic reperfusion damage166,167,168

. On the other

hand, NO and its reaction products have also been

demonstrated to cause detrimental effects on the

reperfused heart169

. The treatment of heart with NO

donors, such as S-nitroso-N-acetylpenicillamine

(SNAP) or 3-morpholinosydnonimine hydrochloride

(SIN-1) increases the formation of ONOO– and

exacerbates the myocardial oxidative damage after

I/R170

. Interaction between NO and superoxide during

the early phase of reperfusion has been demonstrated

to form peroxynitrite, an important determinant of

post-ischemic myocardial function171

. Peroxynitrite

causes cellular damage by lipid peroxidation,

oxidation of sulfhydryl groups and inhibition of

signaling pathways by nitration of tyrosine residues

and DNA strand breaks172-174

. Increased levels of

nitrotyrosine (an indicator of peroxynitrite

production) have also been reported in control heart

subjected to I/R. Heart pretreated with tempol

combined with NO donor NCX-4016 significantly

attenuates the peroxynitrite production (Figure 7)175

.

Increased formation of ROS, following hypoxia-

reoxygenation is associated with low antioxidant

capacity of myocardial tissue. The catalase activity was

reported to be low in myocytes and endothelial cells and

most of it is compartmentalized within peroxisomes.

This subcellular localization prevents catalase from

being an efficient scavenger of H2O2, resulting from

SOD activity in the cytosol176

. Insufficient antioxidant

capacity of tissue to scavenge the increased content of

ROS, following hypoxia/reoxygenation appears to be an

important contributing factor to tissue dysfunction,

restenosis of bypass grafts and post-balloon angioplasty.

These complications may worsen the efficiency of

interventions used in the treatment of coronary artery

disease177

.

The cascade of events associated with I/R injury,

besides free-radical generation includes release of

cytokines and growth factors, leukocyte adhesion,

platelet aggregation, smooth muscle proliferation, and

mechanical injury178

. Studies have suggested that

reperfusion of the ischemic myocardium results in

cardiomyocyte apoptosis and necrosis in human179,180

and in animal models of I/R injury177

. Although

necrosis represents the classic manifestation of

hypoxia-induced cell damage, myocyte apoptosis

appears to be an early event in cardiac I/R injury181

.

The I/R-induced apoptosis is mediated by different

apoptotic signaling cascades that are mediated by free

radicals and oxidative stress182

.

The activation of mitochondria-initiated pathway

plays an important role in the apoptosis in hearts

subjected to I/R180

. Ischemia-reperfusion results in the

release of cytochrome c from the mitochondria and

the activation of caspase-9 in isolated perfused

hearts183

. Several caspase inhibitors have been

demonstrated to attenuate apoptosis in myocardial I/R

injury183-185

. Prolonged reperfusion after ischemia

leads to down-regulation of the antiapoptotic protein

Bcl-2186

. Myocytes lacking the proapoptotic Bax gene

reduces I/R injury through the blocking of necrotic

and apoptotic pathways187

. Recently, involvement of

mitogen activated protein kinases (MAPKs) has been

demonstrated in ischemic injury188,189

. The stress-

induced p38 MAP kinase pathway is activated in

Figure 7—Nitrotyrosine in coronary effluents from isolated

perfused hearts subjected to IR [Nitrotyrosine (an indicator of

peroxynitrite production) was measured using a microplate

fluorimeter with excitation/emission filters 320/410 nm. Upper:

time course of nitrotyrosine formation in coronary effluent from

hearts subjected to IR. Lower: Nitrotyrosine formation at 1 min of

reperfusion. Data are expressed as percentage of pre-ischemic

baseline represented as mean ± SD (n = 3). *p<0.001 vs pre-

ischemia; **p<0.01 vs control (IR) (Adapted from175)]


432

cardiac myocytes exposed to I/R and plays a role in

the induction of apoptosis190-192

. Inhibition of p38

MAPK decreases cardiomyocyte apoptosis and

improves cardiac function after myocardial ischemia

and reperfusion185,193

. Previous studies have

demonstrated that activation of ERK1/2 after

reperfusion is cardioprotective194-196

. The PI3K-Akt

signaling is an important mediator of cell survival and

promotes the survival of cardiomyocytes in vitro and

in vivo. In addition, it protects against acute I/R-injury

in the mouse heart197,198

.

The cytoprotective effects of sodium

orthovanadate, adrenomedullin, vasodilatory peptide,

mexiletine derivative (H-2693) etc. on I/R injury are

reported to be mediated by the activation of Akt199,200

.

C-phycocyanin, a plant-based antioxidant attenuates

I/R-induced injury through antioxidant and

antiapoptotic actions and modulation of p38 MAPK

and ERK1/2161

. An anti-ischemic drug trimetazidine

(TMZ) derivatized with a pyrroline group (TMZ-Ǿ-

NH) significantly protects heart against I/R-mediated

injury by enhancing the pro-survival Akt activity,

without significant effect of p38 MAPK and ERK1/2

(Figure 8)201

. The protective effect of TMZ

derivatives could be due to the combined effects of

anti-ischemic protection by trimetazidine and ROS

scavenging during I/R. ROS have been reported to

induce transcription factor NF-κB202

. Inhibition of

NF-κB by antioxidants further supports a role of ROS

in the activation of NF-κB203

.

Strategies for inhibition of oxidative stress in CVD Despite the evidence for association of increased

oxidative stress with various vascular diseases, using

antioxidant therapy to prevent cardiovascular

diseases has produced mixed results204,205

. Natural

antioxidant α-tocopherol at a dose of either 400 or

800 IU/day causes a significant reduction in the

combined primary end point of cardiovascular death

and non-fatal myocardial infarction206

. In the

antioxidant supplementation in atherosclerosis

prevention (ASAP) study, a combination α-

tocopherol (272 IU/day) and slow-release aspirin has

been shown to significantly decrease carotid intima-

media thickness in hypercholesterolemic males207

.

The vitamin C (359 mg/day) supplementation is also

associated with a significant reduction in non-fatal

and fatal myocardial infarction208

. In contrast, several

antioxidant supplementation studies have not shown

any effect on primary end points of cardiovascular

events204

.

The inability of some of the antioxidants to prevent

CVD may be attributable to several reasons:

(i) ineffectiveness could relate to optimum dose and

type of antioxidants, (ii) complexity of redox

reactions in vivo, (iii) inability to target specific redox

pathways, although some existing drugs exert some of

their effects through redox-depending signaling

pathways, for example, statins, angiotensin-

converting enzyme inhibitors and protein kinase C

inhibitors. Future treatments may need to target redox

pathways in cell, tissue and pathway-specific manner.

Perhaps, the most exciting prospect is the

development of specific targeting strategies to deliver

redox-active molecules to the mitochondrion,

development of specific ROCK inhibitor or agents

which can upregulate Nrf2.

Conclusion With drastic changes in the life style pattern,

increasing number of subjects is at risk of vascular

disease and there is preponderance of evidence for the

association of increased oxidative stress with various

vascular diseases. These cause premature death from

angina, heart attack, stroke, peripheral artery disease,

hypertension, ischemia and thrombosis. The loss of

control of free-radical formation from the

mitochondrion can contribute to the pathology of

CVD through a number of mechanisms including

damage to mtDNA, enzyme degradation, and

apoptosis and thus contribute to human disease.

However, a better understanding of the ROS-

Figure 8—Akt, ERK1/2 and p38 MAPK phosphorylation in heart

subjected to IR [The phosphorylation of Akt, ERK1/2 and p38

MAPK was measured in hearts subjected to ischemia (30 min)

and after 10 min reperfusion , without and with trimetazidine

(TMZ) and trimetazidine derivatives TMZ-NH and TMZ-φ-NH

(50 µM). Phosphorylated Akt, ERK1/2 and p38 MAPK was

detected by Western blot analysis (Adapted from175)


433

dependent signal-transduction mechanism, their

localization and the integration of both ROS-

dependent transcriptional and signaling pathways in

vascular pathophysiology is a pre-requisite for

effective pharmacological interventions of CVD.

Drugs targeting redox-sensitive pathways,

mitochondria-targeted antioxidants, ROCK inhibitors,

iron chelators and agents that induce antioxidant

enzymes have played a modulating role as

cardioprotective agents that have targeted specific

sub-cellular compartments. Another important step

toward future prospect for effective treatment will be

the development of sensitive and specific markers that

can used clinically to assess the oxidative stress

phenotypes that underlie various vascular pathologies.

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Received 4 August 2009; revised 2 November 2009nopr.niscair.res.in/bitstream/123456789/7246/1/IJBB 46(6... · 2010-01-26 · enzymatic and non-enzymatic for prevention of overload